Enzyme inhibition immunoassay

ABSTRACT

The present invention provides a method for the measurement of an analyte in biological samples whereby an uncompetitive inhibitor is coupled to a ligand and utilized in a homogeneous assay. The analyte can be a drug or drug derivative, hormone, polypeptide, or oligonucleotide. The present invention also provides novel compounds, assay reagents and packaged kits useful for performing such measurements.

This application claims benefit of provisional application 60/141,090, filed Jun. 25, 1999.

BACKGROUND OF THE INVENTION

This invention relates generally to the field of measuring an analyte in a liquid medium. More specifically, it relates to an immunoassay for the measurement of an analyte in a biological sample.

Boguslaski, R. C. et al., U.S. Pat. No. 4,134,792 (1979) describe the use of a reversibly binding enzyme modulator as a labeling substance for the detection of an analyte in a liquid medium, and in particular, the use of competitive inhibitors coupled to an analog of the analyte in immunoassays.

Dorn, A. R., U.S. Ser. No. 09/328,741 filed Jun. 9, 1999 describes a method for the enzymatic measurement of mycophenolic acid in a biological sample.

Inosine-5′-monophosphate dehydrogenase (EC 1.1.1.205) catalyzes the NAD-dependent oxidation of inosine-5′-monophosphate (IMP) to xanthosine-5′-monophosphate (XMP), Magasanik, B. et al., J. Biol. Chem. 226:339-350 (1957) and Jackson et al., Nature 256:331-333 (1975). The enzyme follows an ordered Bi-Bi reaction sequence of substrate and cofactor binding and product release. First, IMP binds to IMPDH. This is followed by the binding of the cofactor NAD. The reduced cofactor, NADH, is then released from the product, followed by the product, XMP. This mechanism differs from that of most other known NAD-dependent dehydrogenases, which have either a random order of substrate addition or require NAD to bind before the substrate.

Two isoforms of human IMPDH, designated type I and type II, have been identified and sequenced, Collart et al., J. Biol. Chem. 263:15769-15772 (1988) and Natsumeda et al., J. Biol. Chem. 265:5292-5295 (1990). Each isoform is 514 amino acids, and both isoforms share 84% sequence identity. Both IMPDH type I and type II form active tetramers in solution, with subunit molecular weights of 56 kDa, Yamada et al., Biochemistry 27:2737-2745 (1988).

SUMMARY OF THE INVENTION

In its narrowest application, the present invention is exemplified by a quantitative homogeneous immunoassay specific for mycophenolic acid based upon the specific uncompetitive inhibition of the enzyme inosine-5′-monophosphate dehydrogenase (IMPDH) by mycophenolic acid. IMPDH inhibition depends only upon the concentration of mycophenolic acid due to the uncompetitive nature of inhibition by mycophenolic acid. Thus, the greater the mycophenolic acid concentration, the greater the inhibition of the enzyme. Measuring the formation of reduced nicotinamide adenine dinucleotide (NADH) at 340 nm monitors the reaction. IMPDH catalyzes the following reaction:

Development of a homogeneous immunoassay can be accomplished through the attachment of a ligand to a position on mycophenolic acid or an MPA derivative or any uncompetitive inhibitor of IMPDH where such attachment does not interfere with the uncompetitive inhibition of IMPDH. In the absence of analyte, analyte-specific antibody binds the ligand-MPA derivative and prevents its inhibition of IMPDH. In the presence of analyte, analyte binds to its antibody, thus freeing up ligand-MPA derivative to inhibit IMPDH (Table 3, part C.).

The rate of formation of NADH (reduced nicotinamide adenine dinucleotide) can be measured by monitoring the change in absorption at a wavelength of 340 nm, i.e., the characteristic absorption region of NADH, and this change in absorption can then be correlated to analyte concentration.

Attractive positions for ligand attachment occur on the hexanoic chain of mycophenolic acid and its derivatives, especially at the 4′ carbon and the 5′ carbon (FIG. 1). Several compounds meet these criteria. See Nelson, P. H. et al., Journal of Medicinal Chemistry 33:833-838 (1990), Rohloff, J. C.,et al., Tetrahedron Letters 36 (43):7803-7806 (1995), Artis, D. R. et al., PCT Publication WO 95/22536 (1995) and Artis, D. R. et al., PCT Publication WO 95/22538 (1995). Several of these derivatives show greater inhibition than mycophenolic acid and thus could impart greater sensitivity to a homogeneous immunoassay. See Smith, D. B. et al., J. Org. Chem. 61:2236-2241 (1996).

In a preferred embodiment of the present invention, an uncompetitive inhibitor, mycophenolic acid (MPA) was coupled to a ligand and used in a homogeneous immunoassay for mycophenolic acid.

Use of an uncompetitive inhibitor-ligand conjugate is preferred over a competitive inhibitor-ligand conjugate since the former conjugate is far less susceptible to interferences from drugs and naturally occurring substances, which commonly can be competitive inhibitors of enzymes. Uncompetitive inhibitors of enzymes are rare in nature and should therefore be less susceptible to interferences from drugs and natural substances.

In its broadest application, the present invention can be used to measure any analyte. Specifically exemplified herein is the use of the invention for measurement of mycophenolic acid, digoxigenin, thyroxine and theophylline. Also taught is the use of the invention for measurement of any analyte including drugs or drug derivatives, hormones, polypeptides and oligonucleotides. Examples of other drugs or drug derivatives which would be suitable for assay using the method of the present invention include therapeutic drugs such-as antibiotics, e.g., gentamicin, amikacin, tobramycin, netilmicin and vancomycin; cardiac drugs, e.g., digoxin, digitoxin, N-acetyl procainamide, procainamide, quinidine and lidocaine; anti-seizure drugs, e.g., phenytoin, phenobarbital, primidone, valproic acid, ethosuximide and carbamazepine; analgesics, e.g., acetaminophen and acetylsalicylic acid; and immunosuppressants, e.g., MPA, cyclosporin, rapamycin (sirolimus) and FK506 (tacrolimus), and drugs of abuse such as amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine metabolite (benzoylecgonine), methadone, opiates, phencyclidine, propoxyphene and LSD. Examples of hormones which would be suitable for assay using the method of the present invention include thyroxine, thyroid stimulating hormone, estrogen, progesterone, testosterone, prolactin, follicle stimulating hormone, chorionic gonadotropin and leuteinizing hormone. Examples of polypeptides which would be suitable for assay using the method of the present invention include proteins and epitopes such as hemoglobin Alc, troponin-T and troponin-I. Examples of oligonucleotides which would be suitable for assay using the method of the present invention include specific oligonucleotide sequences that can hybridize under stringent conditions with sequences sufficiently specific for the detection and quantification of gonorrhoeae, human immunodeficiency virus (HIV), chlamydia and hepatitis infections.

Another aspect of the present invention relates to a test kit for conducting an assay for the determination of as analyte via the method of the present invention comprising, in packaged combination, one or more reagent compositions comprising NAD, IMPDH, IMP and a conjugate comprising MPA and a ligand of the analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of mycophenolic acid and the structure of the ligand-inhibitor conjugate, MPA-MPA (bis-MPA).

FIG. 2 is a graph prepared by plotting the results obtained in Example 20 in which samples containing thyroxine were assayed according to the present invention. Concentration of thyroxine is plotted on the X-axis and delta (or end-point) absorbance at 340 nm is plotted on the Y-axis.

FIG. 3 is a graph prepared by plotting the results obtained in Example 23 in which samples containing digoxigenin were assayed according to the present invention. Concentration of digoxigenin is plotted on the X-axis and delta (or end-point) absorbance at 340 nm is plotted on the Y-axis.

FIG. 4 is a graph prepared by plotting the results obtained in Example 26 in which samples containing digoxigenin were assayed according to the present invention. Concentration of digoxigenin is plotted on the X-axis and rate of absorbance at 340 nm is plotted on the Y-axis.

FIG. 5 is a graph prepared by plotting the results obtained in Example 29 in which samples containing theophylline were assayed according to the present invention. Concentration of theophylline is plotted on the X-axis and rate of absorbance at 340 nm is plotted on the Y-axis.

FIG. 6 illustrates the preparation of 5′-isoprenyl[OH]-MPA[OMEM] methyl ester as described in Examples 4, 5, 6 and 7.

FIG. 7 illustrates the preparation of 5′-[isoprenyloxycarbonylaminoethylene-amido)-thyroxine(N-tBoc)]-MPA[OMEM] methyl ester conjugate as described in Examples 8, 9 and 10.

FIG. 8 illustrates the preparation of L-thyroxine(N-tBoc)-amidoethyleneamine as described in Example 9.

FIG. 9 illustrates the preparation of 5′-[(isoprenyloxycarbonylamino-ethyleneamido)-thyroxine]-MPA conjugate as described in Examples 11 and 12.

FIG. 10 illustrates the preparation of 5′-carboxymethyl-MPA t-butyl ester as described in Examples 13, 14 and 15.

FIG. 11 illustrates the preparation of 5′-(digoxigenin-3-yl-β-amidomethyl)-MPA t-butyl ester conjugate as described in Example 16.

FIG. 12 illustrates the preparation of 5′-(digoxigenin-3-yl-β-amidomethyl)-MPA conjugate, isomer A and isomer B, as described in Example 17.

FIGS. 13 and 14 ilustrate the preparation of 4′-(digoxigenin-3-yl)-oxymethylcarbonyl-DADOO-carbonylmethyl]-MPA, as described in Examples 31 and 32.

FIG. 15 illustrates the preparation of 5′-[(theophylline-8-butyramidoethylamino-carbonyloxy)-isoprenyl]-MPA[OMEM] methyl ester conjugate as described in Example 33.

FIG. 16 illustrates the preparation of 5′-[(theophylline-8-butyramidoethylaminocarbonyloxy)-isoprenyl]-MPA as described in Example 35.

FIG. 17 illustrates the preparation of 5′-(succinimido-N-oxy)carbonylmethyl-MPA methyl ester as described in Examples 36, 37, 38 and 39.

FIG. 18 illustrates the preparation of 5′-[(digoxigenin-3-yl)-oxymethylcarbonyl-DADOO-carbonyhnethyl]-MPA as described in Examples 40 and 41.

DETAILED DESCRIPTION OF THE INVENTION

It has been found that there are some properties of mycophenolic acid inhibition of IMPDH that should facilitate the development of homogeneous enzyme immunoassays. The sensitivity (10-20 nM) of mycophenolic acid inhibition and its uncompetitive inhibition favor its use in homogeneous enzyme immunoassays. By carefully coupling a; ligand to mycophenolic acid to form a ligand-inhibitor conjugate, one can develop a homogeneous enzyme immunoassay for a specific analyte. Coupling a ligand to an uncompetitive inhibitor is greatly preferred in the specific case of mycophenolic acid measurement since many competitive inhibitors of IMP and NAD exist and would reduce assay specificity. In general, use of competitive inhibitors in ligand-inhibitor conjugates will lead to interferences in assay performance since many drugs and naturally occurring substances occur as competitive inhibitors, which would greatly reduce specificity.

An uncompetitive inhibitor of IMPDH enzyme is a substance that inhibits by binding at the active site of the enzyme and does not compete with IMP or NAD for inhibition of the enzyme. Uncompetitive inhibition occurs when an inhibitor combines reversibly with the IMPDH-XMP complex at the active site of the enzyme to form IMPDH-XMP-inhibitor complex, which then is unable to release the XMP product. This type of inhibition is not reversed by increasing substrate concentration. NADH is released only once, while XMP is not released. On the other hand, noncompetitive inhibitors bind at a site other than the substrate binding site. Noncompetitive inhibition is also not reversed by increased substrate concentration.

The IMPDH enzyme preferred for use is a recombinant IMPDH-II enzyme from human T lymphocytes.

The sensitivity of the present immunoassay to the analyte concentration may be modified through the use of different forms of IMPDH that vary in sensitivity to inhibition by MPA. For example, the inhibitor constants (K_(i)) of several forms of human IMPDH for MPA are as follows:

K_(i) IMPDH form (IC₅₀) Reference IMPDH-I 37 nM J. Biol. Chem. 268, 27286- 27290 (1993) IMPDH-II 9.5 nM J. Biol. Chem. 268, 27286- 27290 (1993) IMPDH-II ser 275 → ala 275 4 nM Cell 85, 921-930 (1996)

Since the different enzyme forms have varied sensitivities to MPA inhibition, they should also have varied sensitivities to inhibition by ligand-inhibitors and thus have utility in producing immunoassays with differing levels of sensitivity or measuring ranges of analytes.

The invention provides for the adjustment of assay sensitivity. The adjustment or modification of assay sensitivity may not be easily obtainable without the use of different forms of IMPDH of varied sensitivity to MPA or MPA derivatives or any uncompetitive inhibitor of IMPDH.

Sample suspected of containing the analyte: Any sample that is reasonably suspected of containing the analyte, i.e., mycophenolic acid or other IMPDH inhibitor, can be analyzed by the method of the present invention. The sample is typically an aqueous solution such as a body fluid from a host, for example, urine, whole blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus or the like, but preferably is plasma or serum. The sample can be pretreated if desired and can be prepared in any convenient medium that does not interfere with the assay. An aqueous medium is preferred.

Measuring the amount of mycophenolic acid: Quantitative, semi-quantitative and qualitative methods as well as all other methods for determining mycophenolic acid are considered to be methods of measuring the amount of mycophenolic acid. For example, a method that merely detects the presence or absence of mycophenolic acid in a sample suspected of containing mycophenolic acid is considered to be included within the scope of the present invention. The terms “detecting” and “determining”, as well as other common synonyms for measuring, are contemplated within the scope of the present invention.

The determination of MPA according to the present invention may be conducted by a rate-assay method wherein change in absorbance of NADH per unit time is measured or by an end-point method wherein the reaction is quenched after a certain period of time has elapsed. The method c an easily be applied to automated analyzers for laboratory or clinical analysis. Examples of automated laboratory analyzers are COBAS INTEGRA and ROCHE/HITACHI series analyzers (Roche Diagnostics, Indianapolis, Ind.). Other methods for measuring NADH are also contemplated, for example, the reduction of NAD is coupled to the reduction of a tetrazolium salt, 2-p-nitrophenyl-5-phenyl tetrazolium chloride (INT), with phenazine methosulfate serving as an intermediate electron carrier, as described in Babson, A. L. et al, Clinical Chemistry 19(7):766-769 (1973).

Calibration material means any standard or reference material containing a known amount of the analyte to be measured. The sample suspected of containing the analyte and the calibration material are assayed under similar conditions. Analyte concentration is then calculated by comparing the results obtained for the unknown specimen with results obtained for the standard. This is commonly done by constructing a calibration curve such as in FIGS. 2, 3, 4 and 5.

Ancillary materials: Various ancillary materials will frequently be employed in an assay in accordance with the present invention. For example, buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components. Frequently, in addition to these additives, additional proteins may be included, such as albumin, or surfactants, particularly non-ionic surfactants, or the like.

IMPDH refers to the enzyme inosine-5′-monophosphate dehydrogenase, EC 1.1.1.205, which catalyzes the formation of xanthine-5′-monophosphate from inosine-5′-monophosphate. The present invention contemplates the use of IMPDH from natural or recombinant sources or site-directed mutants, and any isoform, site-directed mutant or a mixture of isoforms may be used.

It is to be understood that any reference throughout the specification and claims to mycophenolic acid is meant to cover mycophenolic acid as well as its biologically active and therapeutically active metabolites and derivatives, which behave in a biological sense, i.e. via IMPDH inhibition, as mycophenolic acid.

Another aspect of the present invention relates to kits useful for conveniently performing the assay methods of the invention for the determination of an analyte. To enhance the versatility of the subject invention, reagents useful in the methods of the invention can be provided in packaged combination, in the same or separate containers, in liquid or lyophilized form so that the ratio of the reagents provides for substantial optimization of the method and assay. The reagents may each be in separate containers or various reagents can be combined in one or more containers depending on the cross-reactivity and stability of the reagents.

The kit of the present invention comprises NAD, a ligand-inhibitor conjugate comprising an inhibitor of IMPDH and a ligand of the analyte, IMP and IMPDH. The reagents may remain in liquid form or may be lyophilized. The kit can further comprise calibration and control materials.

However, the present invention has applications beyond the measurement of mycophenolic acid. Using the approach described here, any ligand (such as, but not limited to, theophylline, digoxin, phenytoin, peptide fragments, oligonucleotides, morphine, cocaine etc.) that has been derivatized with an appropriate reactive group for attachment to mycophenolic acid or MPA derivatives or any uncompetitive enzyme inhibitor of IMPDH suitably derivatized at the 4′-position or the 5′-position can be used to measure the corresponding analyte in an unknown sample. Derivatization at these positions creates new optically active centers. Stereoisomers are considered within the scope of the present invention.

A central feature of the present invention is the ability to synthesize MPA derivatives substituted at the 5′-position with a suitable linking group which can be extended to couple to drug derivatives. Two different types of linking groups have been disclosed in this invention, 5′-carboxy alkyl MPA drug derivatives and 5′-isoprenyl linking MPA drug derivatives.

Substitution at the 5′-position of MPA ester involves alkylation reaction using alkyl halo acetate in the presence of sodium bis(trimethylsilyl)amide and DMPU at low temperature. Use of HMPA instead of DMPU appeared to improve the alkylation reactions in general. This alkylation reaction can be extended to use alkyl halo propionate or alkyl halo butyrate to prepare 5′-carboxyethyl MPA ester or 5′-carboxypropyl MPA ester after selective ester hydrolysis at the linker. The carboxy function at the linker then permits a wide variety of synthetic routes by conversion to an active ester and then to couple to suitably derivatized drug derivatives, most commonly, a drug with a linker-containing amino group to generate an amide linkage.

The substitution at the 5′-position of MPA ester using bromo-isoprenyl linker (1) in the presence of sodium bis(trimethylsilyl)amide and HMPA provided MPA 5′-isoprenyl TBDPS derivative. Hydrolysis of TBDPS group provided 5′-isoprenyl[OH]-MPA[OMEM] (5). This key intermediate alcohol has been activated by conversion to N-hydroxysuccinimidyl carbonate by treatment with disuccinimidyl carbonate in the presence of triethyl amine. The activated carbonate was used without isolation to couple to amino modified drugs to provide MPA drug conjugates with a urethane linkage. The urethane linkage can also be produced by use of activation of the alcohol with 1,1′-carbonyldiumidazole or p-nitrophenyl chloroformate and reaction of the intermediates with the amino group modified drug derivatives. This synthesis can be extended to MPA bis-isoprenyl drug conjugates by use of bromo-bis isoprenyl TBDPS. The bromo-bis-isoprenyl TBDPS can be prepared from geraniol derivative following the literature procedure as described for chloro-bis-isoprenyl TBDPS in J. Org. Chem, 62, 3529 (1997).

The inhibition of IMPDH by mycophenolic acid is described by Anderson, J. H. et al., Journal of Biological Chemistry 243(18):4762-4768 (1968). Inhibitors of IMPDH are also described in U.S. Pat. Nos. 5,380,879, 5,444,072, 5,493,030, 5,536,747, 5,633,279 and 5,807,876 and in PCT publications WO 94/01105 and WO 94/12184.

The cloning and expression of human IMPDH in E. coli has been described by Konno, Y. et al., J. Biol. Chem 266(1):506-509 (1991). Collart, F. R. et al., U.S. Pat. No. 5,665,583 (1997) also describe the cloning and expression in E. coli of human IMPDH.

Analyte refers to the substance, or group of substances, whose presence or amount thereof in a liquid medium is to be determined and is meant to include any drug or drug derivative, hormone, protein antigen or oligonucleotide.

Antibody, or preferably, receptor, means a specific binding partner of the analyte and is any substance, or group of substances, which has a specific binding affinity for the ligand to the exclusion of other substances.

Ligand means any substance, or group of substances, which behaves essentially the same as the analyte with respect to binding affinity of the antibody for the analyte and is meant to include any drug or drug derivative and isomers thereof, hormone, polypeptide, or nucleotide.

An example of the measurement of a drug would involve the coupling of morphine thiocyanate or cocaine NHS ester derivatives described by Salamone, S. J. et al., U.S. Pat. No. 5,618,926 (1997) to 4′- or 5′-substituted alkyl amino derivatives of mycophenolic acid or MPA derivatives or any uncompetitive inhibitors of IMPDH to measure morphine or cocaine in unknown samples. Plotting the concentration of drug versus the IMPDH activity can produce a quantitative assay.

The present invention can also be used in molecular diagnostics to determine the presence of a specific target DNA or RNA. In this case a nucleotide-mycophenolic acid conjugate (N-MPA) would be able to inhibit IMPDH when it is not part of an oligonucleotide chain. Once the N-MPA is incorporated into an oligonucleotide chain, the ability of the mycophenolic acid portion of the conjugate to inhibit IMPDH would be modulated.

The N-MPA conjugate can be prepared according to procedures that are well known in the art. The nucleotide can be derivatized through the sugar, base or phosphate moiety in such a way to allow it to be a substrate for enzymes that synthesize oligonucleotides. For example a derivatized nucleotide base could have a tether containing an electrophile or nucleophile. This derivative can then be reacted with an appropriately derivatized MPA molecule to form an N-MPA conjugate.

The assay would be performed by adding a percentage of the N-MPA conjugate to the nucleotide mixture that would be used to synthesize the oligonucleotide. A sample would be added along with the enzymes, and probes used to polymerize the oligonucleotide, DNA and RNA polymerases would be examples of such polymerizing enzymes. At the end of the polymerizing step, IMPDH along with the appropriate IMPDH reactants would be added. If the sample does not contain the target oligonucleotide sequence, then oligonucleotide synthesis would not proceed and the N-MPA would be free to inhibit IMPDH. If the target sequence is present in the sample, then the N-MPA would be incorporated into the oligonucleotide and less N-MPA conjugate would be available to inhibit the IMPDH. The concentration of free N-MPA would be inversely proportional to the IMPDH activity. The more the free N-MPA is depleted, the higher the IMPDH activity will be.

This assay can be used on both a qualitative and quantitative basis to determine the presence of particular target DNA or RNA molecules. The amount of target present in the sample will be proportional to the IMPDH activity.

Modulation of IMPDH activity by MPA derivatives can also be applied to the detection of protein antigens. A short polypeptide which represents the epitope or mimitope of a larger antigen can be derivatized, according to methods known in the art, to contain a group that can be attached to an appropriately derivatized MPA molecule to form a polypeptide-MPA conjugate (PP-MPA). See Amerongen, A. V. et al., Peptide Research 5(5):269-274 (1992).

The assay is performed by mixing a clinical sample with PP-MPA, antibody or appropriate binding partner selective for the antigen of interest, IMPDH and the appropriate reactants used in the IMPDH assay. If the sample does not contain the antigen, the PP-MPA conjugate will be bound by the antibody and will not be available to inhibit IMPDH. If the sample contains the specific target antigen for which the assay is designed, the PP-MPA conjugate will compete with the antigen for the limited antibody present. The amount of inhibition of IMPDH is proportional to the amount of free antigen present in the sample. A quantitative assay can be generated by plotting the concentration of antigen versus the amount of IMPDH activity. The amount of antigen present will be inversely proportional to the amount of IMPDH activity.

A more complete understanding of the present invention will be obtained by reference to the following non-limiting examples.

Abbreviations used:

ACES N-[2-acetamido]-2-aminoethanesulfonic acid DADOO 1,8-diamino-3,6-dioxa-octane 2,2′-(ethylenedioxy)bis-ethylamine DCC 1,3-dicyclohexylcarbodiimide DMF N,N-dimethylformamide DMPU 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone EDC 1-ethyl-3(3-dimethylaminopropyl)carbodiimide HMPA hexamethylphosphoramide MEM 2-methoxyethoxymethyl NaHMDS sodium bis(trimethylsilyl)amide NHS N-hydroxysuccinimide RP-HPLC reverse phase high-performance liquid chromatography TAPSO 3-[N-tris-(hydroxymethyl)methylamino]-2-hydroxy- propanesulfonate, Na TBAF tetrabutylammonium fluoride TBDPS tert-butyldiphenylsilyl tBoc tert-butoxycarbonyl TCEP tris (2-carboxyethyl) phosphine, hydrochloride THF tetrahydrofuran

In the examples that follow, boldface numbers refer to the corresponding structure in the drawings.

EXAMPLE 1 Preparation of Antibody Reagent for Mycophenolic Acid Assay

One liter of an antibody reagent, or first reagent, was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 6.0 grams of ACES were added and dissolved. The pH was adjusted to 6.0 with 2N NaOH. Then 0.53 grams NAD were added and dissolved completely. Next 10 ml ascites anti-MPA monoclonal antibody was added. Then 0.95 grams sodium azide was added and dissolved, and finally 1.0 gram of SUTTOCIDE A (GAF Chemicals Corp.) was added and dissolved. The volume was adjusted to one liter with deionized water.

TABLE 1 Composition of Antibody Reagent Antibody reagent, pH 6.0 Molec. 150 μl Component wt. M g/l Function final M ACES 182.2 0.0330 6.0 Buffer, NAD 0.0163 stabilizer NAD 663.4 0.0008 0.53 Enzyme substrate 0.0004 Anti-MPA — — 1:100 Monoclonal anti- — body Na azide 65.0 0.0146 0.95 Anti-microbial 0.0072 preservative SUTTOCIDE A 127.1 0.004 1.0 Anti-microbial 0.004 (50%) preservative

The antibody used was prepared according to the procedure described by Alexander et al. in PCT patent publication WO 96/02004 (1996). Any antibody with specificity for MPA, either monoclonal or polyclonal, may be used in the practice of the present invention. Monoclonal antibodies are generally preferred, however, because of their ease and consistency of production.

EXAMPLE 2 Preparation of Enzyme Reagent for Mycophenolic Acid Assay

One liter of an enzyme reagent, or second reagent, was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 56.3 grams of NaTAPSO was added and completely dissolved. Then 81.6 grams of sodium acetate-3H₂O were added and dissolved. Next, 1.15 grams of TCEP were added and dissolved. Next 0.319 grams of IMP were added and dissolved, then 2.23 grams of Na₂EDTA were added and dissolved. Next 1.0 gram of SUTTOCIDE A was added and dissolved. The volume was adjusted to one liter with deionized water. Approximately 13.4 ml of recombinant human IMPDH-II were added (E. coli lysate) and dissolved completely in order to achieve a change in absorbance at 340 nm of 4400×10⁻⁴ over 5 minutes at 37 ° C. Next 0.002 grams of ligand-inhibitor conjugate (MPA-MPA, Example 42) were added and dissolved.

The procedure used for cloning and purification of IMPDH-II is described in Carr, S. F. et al., J. Biological Chemistry 268(36):27286-27290 (1993), the content of which is herein incorporated by reference. Cloning of IMPDH is also described in Collart, F. R. et al., U.S. Pat. No. 5,665,583 (1997). IMPDH from natural sources is not commercially available. Any natural source of IMPDH that is inhibited by MPA, MPA-derivative or uncompetitive inhibitor of IMPDH is also suitable.

Effective amount of reagent components are variable depending on specific needs and may be adjusted by simple laboratory experimentation to meet particular assay requirements.

The ligand-inhibitor concentration was chosen based on the degree of IMPDH inhibition by the ligand-inhibitor with and without specific antibody to the ligand. The maximum difference in percent change in inhibition with and without antibody was chosen for use in immunoassay. It was found that in general, a concentration of inhibitor slightly less than its IC₅₀ value and a molar concentration of antibody slightly less than half the molar concentration of ligand-inhibitor produced the maximum difference in percent change in IMPDH inhibition. With two binding sites per antibody, the total number of ligand binding sites was slightly less than the total molar concentration of ligand-inhibitor. The enzyme activity had a slight effect on the IC₅₀ values of the ligand-inhibitor. The lower the enzyme activity, the lower the IC₅₀ value of the ligand-inhibitor. This is consistent with Nowak, I. et.al, Therapeutic Drug Monitoring 19, 358-360 (1997). Therefore, the IMPDH activity was adjusted to produce the lowest IC₅₀ value for the ligand-inhibitor.

TABLE 2 Composition of Enzyme Reagent Enzyme reagent, pH 8.0 Molec. 150 μl Component wt. M g/l Function final M Na TAPSO 281.3 0.2 56.3 Buffer 0.1 Na acetate-3H₂O 136.1 0.6 81.6 Enzyme stabilizer-reduces 0.3 enzyme aggregation TCEP 286.65 0.004 1.15 Enzyme stabilizer-reduces 0.002 cysteine sulfhydryl groups Na₂ IMP 392.2 0.0008 0.319 Enzyme substrate 0.0004 Na₂EDTA 372.2 0.006 2.23 Enzyme stabilizer-chelates 0.003 heavy metals, maintains reduced cysteine sulfhydryls SUTTOCIDE A 127.1 0.004 1.0 Anti-microbial preservative 0.004 (50%) IMPDH-II — — — Enzyme — MPA-MPA 581 3.4 × 10⁻⁶ 0.002 Ligand-inhibitor 1.7 × 10⁻⁶

EXAMPLE 3 Measurement of Mycophenolic Acid

Human plasma samples spiked with mycophenolic acid were assayed for MPA using the method of the present invention. An HITACHI 717 analyzer (Roche Diagnostics Corp., Indianapolis) was programmed to dispense 3 μl of sample and 150 μl of antibody reagent into a 37° C. cuvette, which were then mixed and incubated for 5 minutes after which time 150 μl of enzyme reagent was added and mixed. The difference in absorbance at 340 nm was calculated from the initial addition of enzyme reagent to 5 minutes following enzyme reagent addition.

TABLE 3 IMPDH activity as a function of ligand-inhibitor conjugate, antibody and analyte concentration 3 μl Sample 150 μl Antibody 150 μl Enzyme (MPA Reagent Reagent ΔA × 10⁻⁴ μg/ml) (+/− anti-MPA) (+/− MPA-MPA) @ 340 nm A 0 − − 4338 5 − − 1939 10 − − 541 B 0 − + 1666 5 − + 557 10 − + 214 C 0 +(1:100) + 3109 5 +(1:100) + 2671 10 +(1:100) + 2135 D 0 +(1:1000) + 1957 5 +(1:1000) + 804 10 +(1:1000) + 304 E 0 +(1:100) − 4330 5 +(1:100) − 4075 10 +(1:100) − 3667

Table 3 shows IMPDH activity as a function of ligand-inhibitor conjugate (MPA-MPA), MPA antibody and mycophenolic acid analyte (MPA) concentration. The data indicates that the ligand-inhibitor conjugate possesses useful inhibitory characteristics necessary for a homogeneous immunoassay; that is, the ligand-inhibitor conjugate is bound by both IMPDH and its specific antibody, and this binding is a mutually exclusive event. With sample set A, the reagents contained no antibody or ligand-inhibitor and produced maximum IMPDH activity. With sample set B, the enzyme reagent contained ligand inhibitor and was used to show the effect of ligand inhibitor on IMPDH activity. Sample set C was set up to show the modulation of IMPDH activity by MPA when both antibody and ligand-inhibitor are present. In Sample set D, not enough antibody was present in the antibody reagent to bind MPA or MPA-MPA conjugate and shows the effect of insufficient antibody concentration. In Sample set E, sufficient antibody is present to bind most of the MPA and shows the effect of antibody binding on MPA alone. Compare these results with those results obtained without antibody observed with sample set A.

EXAMPLE 4 Preparation of Bromo-isoprenyl Linker, MPA-methyl Ester and 3-β-amino-Digoxigenin

Bromo-isoprenyl linker (1) was prepared according to the procedure published by Stephanie E. Sen and Gregory J. Ewing in J. Org. Chem. 62, 3529-3536 (1997).

MPA methyl ester (2) was prepared according to the procedure published by William A. Lee, Leo Gu, Andrew R. Miksztal, Nancy Chu, Kwan Leung and Peter H. Nelson in Pharmaceutical Research 7,161-166 (1990).

3-β-Amino-digoxigenin was prepared according to the procedure published in Bioorganic & Medicinal Chemistry Letters 9,771-774 (1999).

Theophylline amine (17) was prepared according to the procedure published in PCT patent application WO 87/07955 (1987).

EXAMPLE 5 Preparation of 5′-isoprenyl[OTBDPS]-MPA Methyl Ester (3)

A solution of 4.02 ml (4.01 mmol) of sodium bis (trimethylsilyl) amide, 1M solution in tetrahydrofuran (THF), was cooled in a dry ice/acetone bath under argon atmosphere. To this reaction mixture was added dropwise a solution of 600 mg (1.79 mmol) of MPA methyl ester (2) in 7 ml of anhydrous THF. The resulting solution was allowed to stir at −78° C. for 30 minutes and a solution of 84 mg (0.46 mmol) of hexamethylphosphoramide (HMPA) in 2 ml of THF was added. The reaction mixture was allowed to stir for 15 minutes at −78° C. and a solution of 840 mg (2.08 mmol) of bromo-isoprenyl linker (1) in 2 ml of THF was added dropwise. The dry ice bath was removed and the reaction was allowed to slowly warm up to room temperature over a period of 20 minutes. The reaction mixture was quenched with 5 ml of saturated ammonium chloride. To the reaction mixture 100 ml of water was added and extracted with 200 ml of ethyl acetate. The organic layer was separated and the aqueous part was re-extracted with ethyl acetate (3×100 ml). The combined organic part was washed with 100 ml of brine, dried (Na₂SO₄) and concentrated. The crude product was purified by silica gel column chromatography using 20% ethyl acetate in hexane to give 501 mg (43 %, 0.76 mmol) of 5′-isoprenyl[OTBDPS]-MPA methyl ester (3).

EXAMPLE 6 Preparation of 5′-isoprenyl[OTBDPS]-MPA[OMEM] Methyl Ester (4)

A solution of 240 mg (0.36 mmol) of 3 in 5 ml of dry dichloromethane was cooled in an ice-bath. To the reaction mixture was added 76 μl (0.433 mmol) of N,N-diisopropylethylamine followed by 42 μl ( 0.36 mmol) of 2-methoxyethoxymethyl chloride. The mixture was allowed to stir in an ice-bath for 1 hour and room temperature for 18 hours. The mixture was diluted with 15 ml of dichloromethane. The organic layer was washed with 3×20 ml of water, dried (Na₂SO₄) and concentrated. The crude product was purified by silica gel column chromatography using 60% hexane in ethyl acetate to give 175 mg (0.23 mmol, 64%) of 4.

EXAMPLE 7 Preparation of 5′-isoprenyl[OH]-MPA[OMEM] Methyl Ester (5)

A solution of 1.09 g (1.46 mmol) of 4 in 40 ml of THF was cooled at 0° C. To this cooled solution was added 4.37 ml (4.35 mmol) of tetrabutylammonium fluoride (1M solution in THF) under argon atmosphere. The reaction mixture was allowed to stir at 0° C. for 1.5 hours and 1 hour at room temperature. The reaction mixture was quenched ith 5 ml of water and was concentrated. The reaction mixture was diluted with 75 ml of dichloromethane and washed with 3×75 ml of water, dried (Na₂SO₄) and concentrated. The crude product was purified by silica gel column chromatography using 80% ethyl acetate in hexane to give 408 mg (0.806 mmol, 55%) of 5.

EXAMPLE 8 Preparation of L-thyroxine[N-tBoc] (6)

To a magnetically stirred mixture of 4.5 g (5.8 mmol) of L-thyroxine, 18 ml DMF, 5.85 ml of 1N NaOH, 495 mg of NaHCO₃ and 18 ml of water. To this suspension was added 1.28 g (5.8 mmol) of di-t-butyl dicarbonate in 18 ml of dimethylformamide. The resulting reaction mixture was allowed to stir for 4 hours and then concentrated under reduced pressure at room temperature. To the residue 45 ml of methanol was added and the undissolved material was filtered off. To the filtrate 1N HCl (20 ml) was added until the precipitation was complete. The solid was filtered and air-dried to give 4.2 g (4.78 mmol, 82%) of L-thyroxine(N-tBoc) (6).

EXAMPLE 9 Preparation of L-thyroxine(N-tBoc)-amidoethyleneamine (7)

To 500 mg (0.57 mmol) of L-thyroxine(N-tBoc) (6) was added 15 ml of anhydrous DMF followed by 149 mg (1.29 mmol) of N-Hydroxysuccinimide and 283 mg 1.47 mmol) of 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide. The mixture was allowed to stir at room temperature 3 hours. This activated ester was used in situ in the following step.

To 5.5 ml of anhydrous pyridine was added 1.5 ml (22 mmol) of ethylenediamine. The mixture was allowed to stir at room temperature for 5 minutes and the previously made solution of thyroxine activated ester prepared in situ was added dropwise to this solution. The mixture was allowed to stir at room temperature for a period of 48 hours. The reaction mixture was concentrated and 100 ml of saturated Na₂CO₃ solution was added. The solid was filtered off and washed with 100 ml of water. This crude solid was purified by column chromatography using 50% chloroform in methanol to give 250 mg (0.27 mmol, 48%) of 7.

EXAMPLE 10 Preparation of 5′-[(isoprenyloxycarbonylaminoethyleneamido)-thyroxine(N-tBoc)]-MPA[OMEM] Methyl Ester Conjugate (8)

To 25 mg (0.049 mmol) of (5) was added 1 ml of THF followed by 12.6 mg (0.046 mmol) of N,N′-disuccinimidyl carbonate and 20 μl of triethylamine. The mixture was allowed to stir at room temperature for 18 hours to give the corresponding N-hydroxysuccinimidyl carbonate.

L-thyroxine-[N-tBoc]-amidoethyleneamine (7, 22 mg,0.023 mmol) was dissolved in 1 ml of anhydrous DMF and 100 μl of triethylamine was added. To this solution was added the previously prepared MPA-N-hydroxysuccinimidyl carbonate solution (generated in situ) dropwise. The reaction was allowed to stir at room temperature for 30 minutes and concentrated in the rotary evaporator. The residue was purified by preparative RP-HPLC (Rainin C-18 (ODS) 21.4 mm×250 mm) using water/acetonitrile gradient system containing 0.1% trifluoroacetic acid. Product containing fractions were combined, acetonitrile was evaporated and the remaining mixture was lyophilized to give 24 mg (0.016 mmol, 34%) of 8.

EXAMPLE 11 Preparation of 5′-[(isoprenyloxycarbonylaminoethyleneamido)-thyroxine(N-tBoc)]-MPA[OMEM] Conjugate (9)

To 22 mg (0.015 mmol) of 5′-[(isoprenyloxycarbonylaminoethyleneamido)-thyroxine(N-tBoc)]-MPA[OMEM] methyl ester (8) was added 2 ml of methanol and a solution of 40 mg (1.6 mmol) of lithium hydroxide in 1 ml of water. The mixture was allowed to stir at room temperature for 2 days and concentrated in the rotary evaporator. To the reaction mixture 2 ml of water and the pH was adjusted to 6 using dilute phosphoric acid. The reaction was concentrated to dryness and 100 ml of methanol was added. The mixture was filtered and the filtrate was concentrated. The residue was purified by preparative RP-HPLC (Rainin C-18 (ODS) 21.4 mm×250 mm) using water/acetonitrile gradient system containing 0.1% trifluoroacetic acid. Product containing fractions were combined, acetonitrile was evaporated and the remaining mixture was lyophilized to give 11 mg (0.007 mmol, 52%) of 9.

EXAMPLE 12 Preparation of 5′-[(isoprenyloxycarbonylaminoethyleneamido)-thyroxine]-MPA conjugate (11)

To 5 mg (0.003 mmol) of 5′-[(isoprenyloxycarbonylaminoethyleneamido)-thyroxine(N-tBoc)]-MPA[OMEM] methyl ester conjugate (9) was added 1.5 ml of dichloromethane and the solution was cooled at 0° C. To this solution was added an ice-cold solution of 200 μl of 50% trifluoroacetic acid in dichloromethane in two portions at an interval of 5 minutes. The mixture was allowed to stir in ice-bath for additional 15 minutes. This was concentrated under reduced pressure. The product formation was monitored by analytical RP-HPLC (C-18, Vydac 4.6 mm×250 mm) using a gradient system of water/acetonitrile containing 0.1% trifluoroacetic acid. The reaction was resubjected to trifluoroacetic acid treatment. The crude reaction mixture was redissolved in 1.5 ml of dichloromethane and the solution was cooled to 0° C. To this solution was added an ice-cold solution of 200 μl of 50% trifluoroacetic acid in dichloromethane in two portions at an interval of 5 minutes. The mixture was allowed to stir at 0° C. for 25 minutes. The reaction mixture was concentrated in the rotary evaporator and then in the high vacuum pump. The product formation was monitored by analytical RP-HPLC (C-18, Vydac 4.6 mm×250 mm) using a gradient system of water/acetonitrile containing 0.1% trifluoroacetic acid. The results indicated that the reaction is a conjugate mixture containing mostly the MEM-deprotected 5′-[(isoprenyloxycarbonylaminoethylene-amido)-thyroxine(N-tBoc)]-MPA (10) with some desired product (11).

The above product was again resubjected to the trifluoroacetic acid treatment. The above reaction mixture was treated with 2 ml of dichloromethane and cooled in an ice-bath. To the mixture was added an ice-cold solution of 600 μl of 50% trifluoroacetic acid in dichloromethane in three portions at an interval of 5 minutes. The mixture was allowed to stir in the ice-bath for additional 25 minutes. The mixture was concentrated under reduced pressure. The crude product was purified by semi-preparative RP-HPLC (C-18, Vydac, 10 mm×250 mm) using a gradient system of water/acetonitrile containing 0.1% trifluoroacetic acid (3 ml/min). Product containing fractions were combined, acetonitrile was evaporated, and the remaining mixture was lyophilized to give 1.8 mg (0.0013 mmol, 45%) of 11.

EXAMPLE 13 Preparation of MPA t-butyl Ester (12)

Mycophenolic acid (860 mg, 2.68 mmol) was dissolved in a solution of 1,4-dioxane containing 700 μl of conc. H₂SO₄. The solution was transferred into a pressure bottle and this was cooled in ice. Isobutylene (20 ml) was poured into the mycophenolic acid solution. The pressure bottle was capped and stirred magnetically very slowly for 2 days at room temperature. The mixture was allowed to cool under dry ice/acetone and the pressure was slowly released. The mixture was allowed to warm up to room temperature and diluted with 150 ml of ethyl acetate. The aqueous part was separated and extracted with 3×100 ml of ethyl acetate. The combined organic layer was washed with 100 ml of water, 100 ml of saturated NaHCO₃ followed by 100 ml of water and dried (Na₂SO₄) and concentrated. The crude product was purified by silica gel column chromatography using 80% hexane in ethyl acetate to give 590 mg (1.63 mmol, 60%) of MPA t-butyl ester (12).

EXAMPLE 14 Preparation of 5′-ethoxycarbonylmethyl-MPA t-butyl Ester (13)

A solution of 3.2 ml (3.19 mmol) of sodium bis (trimethylsilyl)amide (1.0M solution in THF) was cooled in dry ice/acetone bath to −78° C. under argon atmosphere. To this cooled solution was added 0.32 ml (2.64 mmol) of DMPU and allowed to stir at −78° C. for 15 minutes. A solution of 398 mg (1.10 mmol) of MPA t-butyl ester (12) in 5 ml of freshly distilled THF was added dropwise to the reaction mixture. The reaction mixture was allowed to stir at −78° C. for 30 minutes and 0.18 ml (1.61 mmol) of ethyl bromo acetate was added. The resulting mixture was stirred stirred at −40° C. for 3 hours and 0° C. for 1 hour. The reaction was quenched with 10 ml of saturated ammonium chloride and the mixture was allowed to warm up to room temperature. An additional 80 ml of saturated ammonium chloride was added and the water part was extracted with ethyl acetate (3×80 ml). The organic layers were combined, dried (Na₂SO₄) and concentrated. The crude product was purified by silica gel chromatography, first eluting using 80% hexane in ethyl acetate and then 50% ethyl acetate in hexane to give 330 mg of product containing impurities. This was repurified by preparative RP-HPLC (Rainin C-18 (ODS) 21.4 mm×250 mm) using water/acetonitrile gradient system containing 0.1 % trifluoroacetic acid. Product containing fractions were combined, acetonitrile was evaporated and the remaining mixture was lyophilized to give 163 mg (0.35 mmol, 32%) of 13.

EXAMPLE 15 Preparation of 5′-carboxymethyl-MPA t-butyl Ester (14)

A solution of 141 mg (0.30 mmol) of 13 was prepared in 41.1 ml of DMSO. This was diluted with 81 ml of 0.1M potassium phosphate buffer pH 7.0 and 306 μl of esterase (from pig liver, Roche Diagnostics, suspension 10 mg/ml) was added. The reaction mixture was allowed to stir at room temperature for 3 days and filtered. The filtrate was purified using preparative RP-HPLC (WATERS DELTAPAK C-18 50×250 mm, water/acetonitrile-system containing 0.1% trifluoroacetic acid). Product containing fractions were combined, acetonitrile was evaporated and the mixture was lyophilized to give 76 mg (0.17 mmol, 57%) of 5′-carboxymethyl-MPA t-butyl ester (14).

EXAMPLE 16 Preparation of 5′-(digoxigenin-3-yl-β-amidomethyl)-MPA t-butyl Ester Conjugate (15)

5′-Carboxymethyl-MPA t-butyl ester (20 mg, 46 μmol) was dissolved in 193 μl DMF. To this reaction mixture 7.95 mg (69 μmol) of N-hydroxysuccinimide and 11.4 mg (55.2 μmol) of DCC were added. The reaction mixture was allowed to stir at room temperature for 3 hours. An additional 2.65 mg (23 μmol) of N-hydroxysuccinimide and 2.85 mg (13.8 μmol) of DCC dissolved in 55 μl of DMF were added and the reaction mixture was allowed to stir at room temperature for 1 hour. A solution of 23.2 mg (46 μmol) of 3-β-amino-digoxigenin in 63.8 μl (460 μmol) of triethylamine and 120 μl of DMF was added. The reaction mixture was allowed to stir at room temperature for 1 hour and evaporated to give 68 mg of crude product (15). This was used in the next step without further purification.

EXAMPLE 17 Preparation of 5′-(digoxigenin-3-yl-β-amidomethyl)-MPA Conjugate (16)

The crude product of 5′-(digoxigenin-3-yl-β-amidomethyl)-MPA t-butyl ester (15) was dissolved in 3 ml of dichloromethane. To the reaction mixture 3 ml of trifluoroacetic acid was added and the solution was allowed to stir at room temperature for exact 30 minutes. The solution was then evaporated and the residue was purified immediately as follows:

The residue was dissolved in 1.5 ml of methanol and immediately purified using semi-preparative RP-HPLC (Vydac C-18 300 Å 10 μm 22×250 mm, water/acetonitrile-system containing 0.1% trifluoroacetic acid) to give 7.3 mg of isomer A (16A) and 7.4 mg of isomer B (16B).

TABLE 4 Isomer characteristics Isomer A Isomer B RP-HPLC 93% (UV 226 87% (UV 226 nm) (Vydac C-18 5 μm 300 Å 4.6 × nm) 250 mm; Gradient: 0 to 100% Rt = 25.09 min Rt = 25.34 min acetonitrile + 0.1% TFA in 50 minutes) MS ok ok IH-NMR compatible compatible

EXAMPLE 18 Preparation of Antibody Reagent for Thyroxine Assay

One liter of an antibody reagent, or first reagent, was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 6.0 grams of N-[2-acetamido]-2-aminoethanesulfonic acid ACES were added and dissolved. The pH was adjusted to 6.0 with 2N NaOH. Then 0.663 grams NAD were added and dissolved completely. Then 0.95 grams sodium azide was added and dissolved, and finally 1.0 gram of SUTTOCIDE A was added and dissolved. The volume was adjusted to one liter with deionized water. Finally, 10 milligrams of anti-L-thyroxine monoclonal antibody was added.

TABLE 5 Composition of Antibody Reagent Antibody reagent, pH 6.0 150 μl Component Molec. wt. M g/l Function final M ACES 182.2 0.0330 6.0 Buffer, NAD 0.0163 stabilizer NAD 663.4 0.001 0.6634 Enzyme substrate 0.0005 Anti-L- 150,000 — 0.010 Monoclonal anti- — thyroxine body Na azide 65.0 0.0146 0.95 Anti-microbial 0.0072 preservative Suttocide A 127.1 0.004 1.0 Anti-microbial 0.004 (50%) preservative

The antibody used was prepared according to procedures readily available and known to those skilled in the art to which the present invention belongs. Any antibody with specificity for thyroxine, either monoclonal or polyclonal, may be used in the practice of the present invention. Monoclonal antibodies are generally preferred, however, because of their ease and consistency of production.

EXAMPLE 19 Preparation of Eenzyme Reagent for Thyroxine Assay

One liter of an enzyme reagent, or second reagent, was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 168.8 grams of NaTAPSO was added and completely dissolved. Next, 1.15 grams of TCEP was dissolved. Next 31.4 grams of IMP was dissolved, then 2.23 grams of Na₂EDTA were dissolved. Next 1.0 gram of SUTTOCIDE A was dissolved. The volume was adjusted to one liter with deionized water. Enough recombinant human IMPDH-II was added (as a 35% ammonium sulfate precipitate of an E. coli lysate supernatant) and dissolved completely in order to achieve a change in absorbance at 340 nm of 780×10⁻⁴ over 5 minutes at 37° C. Next 0.0005 grams of ligand-inhibitor 5′-[(isoprenyloxycarbonylamino-ethyleneamido)-thyroxine]-MPA conjugate (11) was added and dissolved.

TABLE 6 Composition of Enzyme Reagent Enzyme reagent, pH 8.0 Molec. 150 μl Component wt. M g/l Function final M Na TAPSO 281.3 0.6 168.8 Buffer, enzyme stabilizer-reduces 0.3 enzyme aggregation TCEP 286.65 0.004 1.15 Enzyme stabilizer-reduces cysteine 0.002 sulfhydryl groups Na₂ IMP 392.2 0.08 31.4 Enzyme substrate, enzyme 0.04 stabilizer Na₂EDTA 372.2 0.006 2.23 Enzyme stabilizer-chelates heavy 0.003 metals, maintains reduced cysteine sulfhydryls Suttocide A (50%) 127.1 0.004 1.0 Anti-microbial preservative 0.004 IMPDH-II — — — Enzyme — 5′-[(isoprenyloxycarbonyl- 1363.44 3.7 × 10⁻⁷ 0.0005 Ligand-inhibitor 1.8 × 10⁻⁷ aminoethyleneamido)thyroxine]-MPA

EXAMPLE 20 Measurement of Thyroxine

Saline samples spiked with L-thyroxine were assayed for thyroxine using the method of the present invention. The samples were prepared by suspending L-thyroxine (Sigma Chemical) in saline, dissolving by the dropwise addition of 1N NaOH, and then diluting with saline to achieve final concentrations of 100, 250, 500 and 1000 ng/ml.

An HITACHI 917 analyzer (Roche Diagnostics Corp., Indianapolis) was programmed to dispense 3 μl of sample and 150 μl of antibody reagent into a 37° C. cuvette, which were then mixed and incubated for 5 minutes after which time 150 μl of enzyme reagent was added and mixed. The rate at 340 nm (mA/min) was calculated during the period 3 minutes 13 seconds to 4 minutes 42 seconds (read window 29-34) following enzyme reagent addition. Results obtained are shown in FIG. 2.

EXAMPLE 21 Preparation of Antibody Reagent for Digoxigenin Assay

One liter of an antibody reagent, or first reagent, was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 6.0 grams of ACES were added and dissolved. The pH was adjusted to 6.0 with 2N NaOH. Then 0.53 grams NAD were added and dissolved completely. Then 0.95 grams sodium azide was added and dissolved, and finally 1.0 gram of SUTTOCIDE A was added and dissolved. The volume was adjusted to one liter with deionized water. Finally, 25 milligrams of anti-digoxigenin monoclonal antibody was added.

TABLE 7 Composition of Antibody Reagent Antibody reagent, pH 6.0 150 μl Component Molec. wt. M g/l Function final M ACES 182.2 0.0330 6.0 Buffer, NAD 0.0163 stabilizer NAD 663.4 0.0008 0.53 Enzyme sub- 0.0004 strate Anti-digoxigenin 150,000 0.025 Monoclonal antibody Na azide 65.0 0.0146 0.95 Anti-microbial 0.0072 preservative SUTTOCIDE A 127.1 0.004 1.0 Anti-microbial 0.004 (50%) preservative

The antibody used is commercially available (Roche Cat. No. 1333062). However, any antibody with specificity for digoxigenin, either monoclonal or polyclonal, may be used in the practice of the present invention. Monoclonal antibodies are generally preferred, however, because of their ease and consistency of production.

EXAMPLE 22 Preparation of Enzyme Reagent for Digoxigenin Assay

One liter of an enzyme reagent, or second reagent, was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 56.3 grams of NaTAPSO was added and completely dissolved. Then 81.6 grams of Na, acetate-3H₂O were added and dissolved. Next, 1.15 grams of TCEP was dissolved. Next 0.319 grams of IMP was dissolved, then 2.23 grams of Na₂EDTA were dissolved. Next 1.0 gram of SUTTOCIDE A was dissolved. The volume was adjusted to one liter with deionized water. Enough recombinant human IMPDH-II was added (as a 35% ammonium sulfate precipitate of an E. coli lysate supernatant) and dissolved completely in order to achieve a change in absorbance at 340 nm of 1280×10⁻⁴ over 5 minutes at 37° C. Next 0.001 grams of ligand-inhibitor (4′-[(digoxigenin-3-yl)-oxymethylcarbonyl-DADOO-carbonylmethyl]-MPA or 5′-[(digoxigenin-3-yl)-oxymethylcarbonyl-DADOO-carbonylmethyl]-MPA) was added and dissolved.

TABLE 8 Composition of Enzyme Reagent Enzyme reagent, pH 8.0 Molec. 150 μl Component wt. M g/l Function final M Na TAPSO 281.3 0.2 56.3 Buffer 0.1 Na acetate-3H₂O 136.1 0.6 81.6 Enzyme stabilizer-reduces enzyme 0.3 aggregation TCEP 286.65 0.004 1.15 Enzyme stabilizer-reduces cysteine 0.002 sulfhydryl groups Na₂ IMP 392.2 0.0008 0.319 Enzyme substrate 0.0004 Na₂EDTA 372.2 0.006 2.23 Enzyme stabilizer-chelates heavy 0.003 metals, maintains reduced cysteine sulfhydryls SUTTOCIDE A (50%) 127.1 0.004 1.0 Anti-microbial preservative 0.004 IMPDH-II — — — Enzyme — 4′-[(digoxigenin-3-yl)-oxy-methyl- 939.12 1.07 × 10⁻⁶ 0.001 Ligand-inhibitor 5.3 × 10⁻⁷ carbonyl-DADOO-carbonylmethyl]-MPA 5′-[(digoxigenin-3-yl)-oxy-methyl- 939.12 1.07 × 10⁻⁶ 0.001 Ligand-inhibitor 5.3 × 10⁻⁷ carbonyl-DADOO-carbonylmethyl]-MPA

EXAMPLE 23 Measurement of Digoxigenin

Saline samples spiked with digoxigenin were assayed for digoxigenin using the method of the present invention. The digoxigenin (Sigma chemical) was first dissolved in 100% DMSO and then added to normal human plasma to achieve concentrations of 0.5, 1.0, 5.0 and 10.0 μg/ml. An HITACHI 717 analyzer (Roche Diagnostics Corp., Indianapolis) was programmed to dispense 20 μl of sample and 150 μl of antibody reagent into a 37° C. cuvette, which were then mixed and incubated for 5 minutes after which time 150 μl of enzyme reagent was added and mixed. The difference in absorbance at 340 nm was calculated from the initial addition of enzyme reagent to 5 minutes following enzyme reagent addition. Results obtained are shown in FIG. 3.

This example shows that conjugates prepared at either the 4′ or 5′ position are useable in the present invention.

EXAMPLE 24 Preparation of Antibody Reagent for Digoxigenin Assay

One liter of an antibody reagent, or first reagent, was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 6.0 grams of ACES were added and dissolved. The pH was adjusted to 6.0 with 2N NaOH. Then 0.663 grams NAD were added and dissolved completely. Then 0.95 grams sodium azide was added and dissolved, and finally 1.0 gram of SUTTOCIDE A was added and dissolved. The volume was adjusted to one liter with deionized water. Finally, 10 or 15 milligrams of anti-digitoxin monoclonal antibody were added.

TABLE 9 Composition of Antibody Reagent Antibody reagent, pH 6.0 150 μl Component Molec. wt. M g/l Function final M ACES 182.2 0.0330 6.0 Buffer, NAD 0.0163 stabilizer NAD 663.4 0.001 0.6634 Enzyme sub- 0.0005 strate Anti-digitoxin 150,000 — 0.010 or Monoclonal — 0.015 antibody Na azide 65.0 0.0146 0.95 Anti-microbial 0.0072 preservative SUTTOCIDE 127.1 0.004 1.0 Anti-microbial 0.004 A (50%) preservative

Less monoclonal antibody was used with digoxigenin isomer B because better assay sensitivity with this inhibitor was observed with lower monoclonal antibody. This may be due to improved competition between IMPDH binding and monoclonal antibody binding with this inhibitor. This could result from an increase in affinity of the inhibitor for IMPDH and monoclonal antibody.

EXAMPLE 25 Preparation of Enzyme Reagent for Digoxigenin Assay

One liter of an enzyme reagent, or second reagent, was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 168.8 grams of NaTAPSO was added and completely dissolved. Next, 1.15 grams of TCEP was dissolved. Next 31.4 grams of IMP was dissolved, then 2.23 grams of Na₂EDTA were dissolved. Next 1.0 gram of SUTTOCIDE A was dissolved. The volume was adjusted to one liter with deionized water. Enough recombinant human IMPDH-II was added (as a 35% ammonium sulfate precipitate of an E. coli lysate supernatant) and dissolved completely in order to achieve a change in absorbance at 340 nm of 3300×10⁻⁴ over 5 minutes at 37° C. Next 0.0002 grams of ligand-inhibitor (5′-[(digoxigenin-3-yl)-β-amdomethyl]-MPA, isomer A or 5′-[(digoxigenin-3-yl)-β-amidomethyl]-MPA, isomer B) was added and dissolved. Or alternatively 0.0005 grams of ligand-inhibitor (5′-[(digoxigenin-3-yl)-oxymethylcarbonyl-DADOO-carbonylmethyl]-MPA, racemic) was added and dissolved.

TABLE 10 Composition of Enzyme Reagent Enzyme reagent, pH 8.0 Molec. 150 μl Component wt. M g/l Function final M NaTAPSO 281.3 0.6 168.8 Buffer, enzyme stabilizer- 0.3 reduces enzyme aggregation TCEP 286.65 0.004 1.15 Enzyme stabilizer-reduces 0.002 cysteine sulfhydryl groups Na₂IMP 392.2 0.08 31.4 Enzyme substrate, enzyme 0.04 stabilizer Na₂EDTA 372.2 0.006 2.23 Enzyme stabilizer-chelates 0.003 heavy metals, maintains reduced cysteine sulfhydryls SUTTOCIDE A (50%) 127.1 0.004 1.0 Anti-microbial preservative 0.004 IMPDH-II — — — Enzyme — 5′-[(digoxigenin-3-yl)-β-amido- 749.91 2.7 × 10⁻⁷ 0.0002 Ligand-inhibitor 1.3 × 10⁻⁷ methyl]-MPA (isomer A) 5′-[(digoxigenin-3-yl)-β-amido- 749.91 2.7 × 10⁻⁷ 0.0002 Ligand-inhibitor 1.3 × 10⁻⁷ methyl]-MPA (isomer B) 5′-[(digoxigenin-3-yl)-oxy- 939.12 5.3 × 10⁻⁷ 0.0005 Ligand-inhibitor 2.7 × 10⁻⁷ methylcarbonyl-DADOO- carbonylmethyl]-MPA (racemic)

EXAMPLE 26 Measurement of Digoxigenin

Saline samples spiked with digoxigenin were assayed for digoxigenin using the method of the present invention. The digoxigenin (Sigma chemical) was first dissolved in 100% DMSO and then added to saline to achieve concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/ml. An HITACHI 917 analyzer (Roche Diagnostics Corp., Indianapolis) was programmed to dispense 3 μl of sample and 150 μl of antibody reagent into a 37° C. cuvette, which were then mixed and incubated for 5 minutes after which time 150 μl of enzyme reagent was added and mixed. The difference in absorbance at 340 nm was calculated from the initial addition of enzyme reagent to 4 minutes 42 seconds following enzyme reagent addition. Results obtained are shown in FIG. 4.

This example illustrates the use of different lengths of linkers in the ligand-inhibitor conjugate.

EXAMPLE 27 Preparation of First Reagent for Theophylline Assay

One liter of a first reagent was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 6.0 grams of ACES were added and dissolved. The pH was adjusted to 6.0 with 2N NaOH. Then 0.66 grams NAD were added and dissolved completely. Then 0.95 grams sodium azide was added and dissolved, and finally 1.0 gram of SUTTOCIDE A was added and dissolved. The volume was adjusted to one liter with deionized water.

TABLE 11 Composition of First Reagent First reagent, pH 6.0 Molec. 150 μl Component wt. M g/l Function final M ACES 182.2 0.0330 6.0 Buffer, NAD 0.0163 stabilizer NAD 663.4 0.0010 0.66 Enzyme substrate 0.0005 Na azide 65.0 0.0146 0.95 Anti-microbial 0.0072 preservative SUTTOCIDE A 127.1 0.004 1.0 Anti-microbial 0.004 (50%) preservative

EXAMPLE 28 Preparation of Second Reagent for Theophylline Assay

One liter of a second reagent was prepared as follows. Approximately 800 ml deionized water was dispensed into a container and 168.8 grams of NaTAPSO were added and completely dissolved. Next, 1.15 grams of TCEP were dissolved. Next 31.4 grams of IMP were dissolved, and then 2.23 grams of Na₂EDTA were dissolved. Next 1.0 gram of SUTTOCIDE A was dissolved. The volume was adjusted to one liter with deionized water. Enough recombinant human IMPDH-II was added (as a 35% ammonium sulfate precipitate of an E. coli lysate supernatant) and dissolved completely in order to achieve a change in absorbance at 340 nm of 3100×10⁻⁴ over 5 minutes at 37° C. Next 0.00027 grams of ligand-inhibitor 5′-[(theophylline-8-butyramidoethylaminocarbonyloxy)isoprenyl]-MPA (20) were added and dissolved. Finally, 10 or 25 milligrams of anti-theophylline monoclonal antibody were added.

TABLE 12 Composition of Second Reagent Second reagent, pH 8.0 150 μl Component Molec. wt. M g/l Function final M Na TAPSO 281.3 0.6 168.8 Buffer, enzyme stabilizer-reduces 0.3 enzyme aggregation TCEP 286.65 0.004 1.15 Enzyme stabilizer-reduces cysteine 0.002 sulfhydryl groups Na₂ IMP 392.2 0.08 31.4 Enzyme substrate, enzyme 31.4 stabilizer Na₂EDTA 372.2 0.006 2.23 Enzyme stabilizer-chelates heavy 0.003 metals, maintains reduced cysteine sulfhydryls SUTTOCIDE A (50%) 127.1 0.004 1.0 Anti-microbial preservative 0.004 IMPDH-II — — — Enzyme — Anti-Theophylline 150,000 — 0.010-0.025 Monoclonal antibody — 5′-[(theophylline-8-butyramido- 738.78 3.6 × 10⁻⁷ 2.7 × 10⁻⁴ Ligand-inhibitor 1.8 × 10⁻⁷ ethylaminocarbonyl-oxy)iso- prenyl]-MPA

The antibody used was prepared according to procedures readily available and known to those skilled in the art to which the present invention belongs. Any antibody with specificity for theophylline, either monoclonal or polyclonal, may be used in the practice of the present invention. Monoclonal antibodies are generally preferred, however, because of their ease and consistency of production.

EXAMPLE 29 Measurement of Theophylline

Saline samples spiked with theophylline were assayed using the method of the present invention. Theophylline (Sigma Chemical) was first dissolved in saline (1 mg/10 ml) and then diluted to achieve concentrations of 0, 5, 10, 20, 40 and 50 μg/ml. An HITACHI 717 analyzer (Roche Diagnostics Corp., Indianapolis) was programmed to dispense 3 μl of sample and 150 μl of first reagent into a 37° C. cuvette, which were then mixed and incubated for 5 minutes, after which time 150 μl of second reagent was added and mixed. The difference in absorbance at 340 nm was calculated from the initial addition of the second reagent to 5 minutes following the second reagent addition. Results obtained are shown in FIG. 5.

In this example, inhibitor and antibody were combined together in the same reagent. The particular theophylline antibody used in this example had a relatively low affinity for the inhibitor compared to that for the drug, so the antibody and inhibitor were combined first before adding sample in order to pre-form the inhibitor-antibody conjugate. Then when the sample containing drug is added, the drug displaces the inhibitor, because of the higher affinity of the antibody for the drug, thereby freeing the inhibitor for inhibiting the enzyme.

EXAMPLE 30 Inhibition Constants of Ligand Inhibitors

The IC₅₀, inhibitor constant, is the molar concentration of inhibitor that produces 50% inhibition of enzymatic activity. This example is used to illustrate potency of enzyme inhibition by various MPA derivatives.

This assay measures the formation of NADH during the conversion of IMP to XMP by IMPDH II. The reaction is monitored using a spectrophotometer at 340 nm for 10 minutes 40° C. The procedure used in this example is that described by Nelson, P. H. et al., J. Med. Chem. 39, 4181-4196 (1996). Inhibitors were diluted in 100% DMSO.

A reaction buffer, 100 ml, was prepared containing 125 mM Tris HCl, pH 8.0, 125 mM KCl, 3.75 mM EDTA, 125 g/ml BSA, 0.0625 mM IMP and 0.125 mM NAD. An enzyme diluent, 100 ml, was prepared containing 100 mM Tris HCl, pH 8.0, 100 mM KCl, 3.0 mM EDTA and 100/ml BSA.

The IMPDH II enzyme was diluted to 0.0065 U/ml in the enzyme diluent. The final concentration of the enzyme in the reaction cuvette was 0.00065 U/ml. Samples (derivatives) were dissolved in DMSO to 1 mg/ml. The samples were further diluted in 100% DMSO. All dilutions were prepared fresh from 1 mM stocks prepared previously.

The assay format was as follows: 800 μl of reaction buffer were added to 6 cuvettes in a spectrophotometer. 100 μl of DMSO only (control) or sample concentrations were added per conjugate/MPA derivative tested. Cuvettes with reaction buffer and sample were mixed by inverting. Cuvettes with reaction buffer and sample were allowed to incubate for 5 minutes at 40° C. Reaction was initiated with the addition of 100 μl of enzyme solution. Cuvettes were mixed again by inverting. The reactions were monitored at 340 nm and data collected for 10 minutes. The results were used to determine the IC₅₀s for the samples.

MPA-isoprenyl (racemic, 3A), was prepared by lithium hydroxide hydrolysis of the ester group and deprotection of the TBDPS group of compound (3) following methods known to those skilled in the art. The chiral MPA-isoprenyl (3B), R-isomer, was made by alkylation of the [(S)-4-benzyl-2-oxazolidinon-1-yl] imide analog [Rohloff et al., Tetrahedron Lett., 36, 7803-7806 (1995)] of (2), at the 5′-position, with the alkyl bromide (1) in a similar manner to that described for the synthesis of (3); followed by deprotection of the TBDPS group and hydrolysis of the oxazolidinyl group using methods known to practitioners of the art.

TABLE 13 Inhibitor constants MPA Derivatives IC₅₀ MPA 2.6 × 10⁻⁸M MPA-MPA 3.2 × 10⁻⁸M MPA-isoprenyl (chiral, R enantiomer, 1.3 × 10⁻⁸M 3B) MPA-isoprenyl (racemic, 3A) 4.8 × 10⁻⁸M 5′-(digoxigenin-3-yl-β-amidomethyl)- 6.8 × 10⁻⁸M MPA (isomer B, chiral, possibly R enantiomer, 16B) 5′-(digoxigenin-3-yl-β-amidomethyl)- 1.7 × 10⁻⁷M MPA (isomer A, chiral, possibly S enantiomer, 16A) 5′-[(digoxigenin-3-yl)-oxymethyl- 1.7 × 10⁻⁷M carbonyl-DADOO-carbonylmethyl]- MPA (racemic, 26) 5′-[(isoprenyloxycarbonylamino- 8.3 × 10⁻⁸M* ethyleneamido)-thyroxine]-MPA (11) 5′-[(theophylline-8-butyramidoethyl- 2.5 × 10⁻⁷M* aminocarbonyl-oxy)isoprenyl]-MPA (20)

This table shows the IC₅₀ data obtained for several MPA derivatives. The IC₅₀ data is the molar concentration of MPA-inhibitor that reduces the enzyme activity by 50%. The IC₅₀ values are determined by using a non-linear curve fit of the plot of fractional activity (enzyme rate with MPA-inhibitor/enzyme rate without any MPA-inhibitor) on the y-axis vs. molar concentration of the MPA-inhibitor in the assay mixture on the x-axis. Sigma-Plot 4.0 software was used for the non-linear curve fit using the regression formula; hyperbolic decay, 2 parameters, y=ab/(b+x), where y=fractional activity, x=molar concentration of inhibitor, a=1, and b=IC₅₀.

The IC₅₀ results show the importance of chirality at the 5′ position of MPA derivative inhibitors of IMPDH, and the spatial geometry of our more potent enantiomers are consistent with Smith, D. B. et.al., Journal of Organic Chemistry 61, 2236-2241 (1996).

The MPA-MPA derivative was used as a prototype inhibitor that had a drug (MPA) attached to the MPA parent structure. Since it was demonstrated that MPA-MPA as a prototype drug-inhibitor worked in an immunoassay for MPA, a measure of the IC₅₀ value for this inhibitor may be a predictor of success in an immunoassay not only for MPA but also for new drugs. Those drug inhibitors with lower IC₅₀ values would be more potent inhibitors of IMPDH activity and thus useful in the described immunoassay. For example, MPA-thyroxine isoprenyl had a lower IC₅₀ value and showed greater sensitivity in an immunoassay than MPA-theophylline. Also, chiral derivatives are more potent inhibitors of IMPDH consistent with a previous publication which reports the S enantiomer of an MPA derivative to be more potent. J. Org. Chem. 61, 2236-2241, (1996).

*The IC5₀ values for these derivatives were determined using a slightly different method. For these derivatives the enzyme rate with and without inhibitor was determined as described in Example 20 for thyroxine and Example 29 for theophylline, except no antibody was added to the second reagent and only saline was used as a sample. Multiple second reagents each containing a different concentration of inhibitor were assayed using an HITACHI 917. The first reagent was as described in Examples 20 and 29 without modification. The fractional rates obtained with these different concentrations of inhibitor were plotted vs. the molar concentration of the inhibitor and the IC₅₀ calculated as described.

EXAMPLE 31 Synthesis of (digoxigenin-3-yl)-oxymethylcarbonyl-DADOO (24)

1.35 g (9.2 mmol) diamino-dioxa-octane were dissolved in 1.3 ml triethylamine and 15 ml dioxane and stirred at room temperature. To this mixture a solution of 500 mg (0.92 mmol) digoxigenin-3-cme-NHS in 10 ml dioxane was added by a dropping funnel within 15 min. After 1 hour stirring at room temperature the solvent was evaporated and the crude product was dissolved in 10 ml methanol and purified with prep. RP-HPLC (Waters Delta Pak C-18 50×250 mm, water/acetonitrile/0.1% TFA). The pure fractions are pooled and lyophilized to give 520 mg oily product.

EXAMPLE 32 Synthesis of [(digoxigenin-3-yl)-oxymethylcarbonyl-DADOO-carbonylmethyl]-MPA (28)

To a solution of 41 mg (0.1084 mmol) dicarboxy MPA (27, Example 43) in 820 μl THF 8.77 μl (0.1084 mmol) pyridine and 15.3 μl trifluoroacetic anhydride were added and the mixture was stirred for 3 hours. 69.3 mg (0.1084 mmol) (digoxigenin-3-yl)-oxymethylcarbonyl-DADOO dissolved in 820 μl pyridine were added and the mixture was stirred 16 hours at room temperature. The reaction mixture was diluted with 1 ml acetic acid and purified using preparative RP-HPLC (Waters Delta Pak C-18 50×250 mm, water/acetonitrile/0.1% TFA). Product containing fractions were pooled and lyophilized to give 66 mg. The product was characterized by ¹H-NMR and MS.

EXAMPLE 33 Preparation of 5′-[(theophylline-8-butyramidoethylaminocarbonyloxy)-isoprenyl]-MPA[OMEM] Methyl Ester Conjugate (18)

To 23 mg (0.045 mmol) of 5′-isoprenyl[OH]-MPA[OMEM] methyl ester (5) was added 1.5 ml of THF followed by 20 mg (0.078 mmol) of N,N′-disuccinimidyl carbonate and 20 μl of triethylamine. The mixture was allowed to stir at room temperature for 18 hours to give corresponding N-hydroxysuccinimidyl carbonate.

Theophylline amine (17), 14 mg (0.045 mmol), was dissolved in 1 ml of anhydrous DMF and 100 μl of triethylamine was added. To this solution was added the previously prepared MPA-N-hydroxysuccinimidyl carbonate solution (generated in situ) dropwise. The solution was purified by preparative RP-HPLC (Rainin C-18 (ODS) 21.4 mm×250 mm) using water/acetonitrile gradient system containing 0.1% trifluoroacetic acid. Product containing fractions were combined, acetonitrile was evaporated and the remaining mixture was lyophilized to give 10 mg (0.011 mmol, 26%) of 18.

EXAMPLE 34 Preparation of 5′-[(theophylline-8-butyramidoethylaminocarbonyloxy)-isoprenyl]-MPA[OMEM] Conjugate (19)

To a solution of 11.7 mg (0.014 mmol) of 5′-[(theophylline-8-butyramidoethylaminocarbonyloxy)isoprenyl]-MPA[OMEM] methyl ester conjugate (18) in 2.4 ml of methanol stirring under argon was added a solution of 24 mg (0.572 mmol) of lithium hydroxide monohydrate in 1.2 ml of water. The reaction was stirred at room temperature under argon, monitoring by RP-HPLC (Vydac C-18; 300 Å 218TP54; 4.6 mm×250 mm; 0.1% TFA-H₂O/0.1% TFA-CH₃CN gradient). After 4 days at room temperature consumption of starting material was almost complete, with increasing appearance of a peak at shorter retention time. The reaction mixture was acidified with dilute phosphoric acid (precipitate), concentrated to dryness, triturated with methanol and the solids filtered off. The filtrate was concentrated to dryness, the residue redissolved in CH₃CN-H₂O (1:1) and purified by preparative RP-HPLC (Rainin C-18 (ODS) 21.4 mm×250 mm) using a 0.1% TFA-H₂O/0.1% TFA-CH₃CN gradient. Fractions containing product were combined, acetonitrile evaporated off under reduced pressure and the residue lyophilized to give 8 mg (0.0097 mmol, 69%) of (19) as a white solid.

EXAMPLE 35 Preparation of 5′-[(theophylline-8-butyramidoethylaminocarbonyloxy)-isoprenyl]-MPA (20)

To a solution of 1.0 mg (0.0012 mmol) of 5′-[(theophylline-8-butyramidoethylaminocarbonyloxy)isoprenyl]-MPA[OMEM] conjugate (19) in 200 μl of dry methylene chloride under argon and cooled in an ice-bath was added a chilled solution of 20 μl of trifluoroacetic acid dissolved in 40 μl of methylene chloride in 3 portions over about a minute. After stirring for 20 minutes while maintaining cooling, volatile material was evaporated off by direct aspiration under high vacuum. RP-HPLC (Vydac C-18; 300 Å 218TP54; 4.6 mm×250 mm; 0.1% TFA-H₂O/0.1% TFA-CH₃CN gradient) and ¹H-NMR (CD₃OD) of the residue indicated partial formation of the desired MEM-deprotected product. The material was re-pooled, concentrated to dryness, redissolved in dry methylene chloride and re-evaporated (3×). The residue was resubjected to the TFA treatment as described above but with stirring for 35 minutes. Solvents were removed as before. RP-HPLC and ¹H-NMR analysis of the residue indicated further formation of desired product and reduction of starting material. The repooled material was re-concentrated, treated as before and resubjected to the TFA treatment as described above for a third time, with stirring for 30 minutes. RP-HPLC analysis of the resulting reaction mixture indicated essential completeness of the reaction, with increases in by-product peaks. The reaction was concentrated to dryness, redissolved in CH₃CN and purified by semi-preparative RP-HPLC (Vydac C-18; 300 Å 218TP510; 0.1% TFA-H₂O/0.1% TFA-CH₃CN gradient). The fractions containing product were pooled, CH₃CN evaporated off under reduced pressure and the residue lyophilized to give about 0.5g (˜0.00068 mmol, ˜56%) of (20) as a white solid.

EXAMPLE 36 Preparation of 5′-t-butoxycarbonylmethyl-MPA Methyl Ester (21)

A solution of 26 ml (26 mmol) of sodium bis(trimethylsilyl)amide (1.0M solution in THF) was cooled in dry ice/acetone bath to −78° C. under argon atmosphere. To this cooled solution was added 2.6 ml (22 mmol) of DMPU and allowed to stir at −78° C. for 15 minutes. A solution of 2.86 g (8.56 mmol) of MPA methyl ester (2) in 45 ml of freshly distilled THF was added dropwise to the reaction mixture. The reaction mixture was allowed to stir at −78° C. for 1 hour and the color of the reaction mixture was turned from pale yellow to yellow-orange. To the reaction mixture was added 1.9 ml (912.8 mmol) of t-butyl bromoacetate and the reaction mixture was allowed to stir at −78° C. for 3 hours. The reaction was quenched with 20 ml of saturated ammonium chloride solution and the mixture was allowed to warm up to room temperature. An additional 200 ml of saturated ammonium chloride was added and the reaction mixture was extracted with 3×200 ml of ethyl acetate. The combined organic layer was washed with 300 ml of saturated ammonium chloride, dried and concentrated. The crude product was purified by silica gel column chromatography using 30% hexane in ethyl acetate to give 3.87 g of a semi-solid. A portion of this product (1.45 g) was purified by RP-HPLC (Rainin C-18 (ODS) 21.4 mm×250 mm) using a gradient system of acetonitrile/water containing 0.1% of trifluoroacetic acid in several runs. Product containing fractions were combined and acetonitrile was evaporated. The residue was lyophilized to give 669 mg (1.49 mmol, 47%) of 5′-t-butoxycarbonylmethyl-MPA methyl ester (21).

EXAMPLE 37 Preparation of 5′-carboxymethyl-MPA Methyl Ester (22)

To 300 mg (0.67 mmol) of 5′-t-butoxycarbonylmethyl-MPA methyl ester (21) was added 15 ml of a solution of 50% trifluoroacetic acid in dichloromethane. The mixture was allowed to stir at room temperature and concentrated. The residue was purified by silica gel column chromatography using 20% methanol in ethyl acetate to give 250 mg (0.63 mmol, 95%) of 5′-carboxymethyl-MPA methyl ester (22).

EXAMPLE 38 t-Butylester Cleavage (alternate method)

To a solution of 360 mg (0.8 mmol) of (21) in 3.6 ml dichloromethane 3.6 ml TFA were added and the reaction mixture was stirred for 10 minutes. 200 ml 0.5M potassium phosphate buffer pH 7.0 was added and the mixture was extracted three times with 100 ml ethyl acetate. The organic layers were pooled, dried over magnesium sulfate and the solvent was evaporated. The product was characterized with HPLC and ¹H-NMR.

EXAMPLE 39 Preparation of 5′-(succinimido-N-oxy)carbonylmethyl-MPA Methyl Ester (23)

To 200 mg crude 5′-carboxymethyl-MPA methyl ester (22), 244 mg (2.12 mmol) N-hydroxysuccinimide and 437 mg (2.12 mmol) dicyclohexylcarbodiimide dissolved in 2 ml THF were stirred for 2 hours at room temperature. The precipitated dicyclohexylurea was filtered off and the filtrate was purified using preparative RP-HPLC (Waters Delta Pak C-18 50×250 mm, water/acetonitrile/0.1% TFA).

Product containing fractions were pooled and immediately lyophilized to give 129 mg of (23). The product was characterized by ¹H-NMR and MS.

EXAMPLE 40 Preparation of 5′-[(dimoxigenin-3-yl)-oxymethylcarbonyl-DADOO-carbonylmethyl]-MPA Methyl Ester (25)

To a solution of 166 mg (0.259 mmol) (digoxigenin-3-yl)-oxymethylcarbonyl-DADOO in 3.48 ml anhydrous DMF and 179 μl triethylamine, 127 mg (0.259 mmol) of (23) in 1.74 ml anhydrous DMF were pipetted. The reaction mixture was stirred for 2 hours. Then 500 μl acetic acid were added to the solution and the mixture was purified using preparative RP-HPLC (Waters Delta Pak C-18 50×250 mm, water/acetonitrile/0.1% TFA). Product containing fractions were pooled and lyophilized to give 240 mg of (25). The product was characterized by ¹H-NMR and MS.

EXAMPLE 41 Preparation of 5′-[(digoxigenin-3-yl)-oxymethylcarbonyl-DADOO-carbonylmethyl]-MPA (26)

200 mg (0.2098 mmol) 5′-[(digoxigenin-3-yl)-oxymethylcarbonyl-DADOO-carbonylmethyl]-MPA methyl ester (25) dissolved in 28.7 ml DMSO were mixed slowly with 56 ml 0.1M potassium phosphate buffer pH 7.0. The mixture became slightly turbid. 219 μl esterase (Roche Diagnostics, Cat. No. 104698) were added and the mixture was stirred for 13 days. The precipitate was filtered and the filtrate was purified using preparative RP-HPLC (Waters Delta Pak C-18 50×250 mm, water/acetonitrile/0.1% TFA). Product containing fractions were pooled and lyophilized to give 68 mg of (26). The product was characterized by ¹H-NMR and MS.

EXAMPLE 42 Synthesis of MPA-MPA

A solution of mycophenolic acid methyl ester (2, 1.337 g, 4 mmol) in 6 ml tetrahydrofuran under inert atmosphere was cooled to −78° C. and treated with sodium hexamethyldisilazide (9 ml, 1M in tetrahydrofuran, 9 mmol) over 25 minutes. After 15 minutes stirring, (E)-4-1,3-dihydro-6-methoxy-4-methoxyethoxymethoxy-7-methyl-3-oxoisobenzofuran-5-yl)-2-methylbut-2-enyl bromide prepared as in U.S. Pat. No. 5,493,030 example ZA-12B (1.36 g, 3.17 mmol) was added as a solution in tetrahydrofuran (4 ml) over 15 minutes. The reaction was allowed to warm slowly to about −40° C. over 1.5 hours, then the reaction was quenched by pouring onto a mixture of ice, water, and concentrated hydrochloric acid (2 ml). Extraction of the product into ethyl acetate (2×100 ml) was followed by drying over magnesium sulfate. The organics were filtered and solvent removed using a rotary evaporator. Chromatography using 40% ethyl acetate in hexanes, then 60% ethyl acetate in hexanes, and finally 80% ethyl acetate in hexanes furnished the alkylation product as an oil (191 mg) which was used in the next reaction.

The alkylation product (190 mg) was dissolved in methanol (30 ml) and treated with 660 mg of p-toluenesulfonic acid and stirred under nitrogen at ambient temperature for 5 hours. The methanol was then removed under reduced pressure and the reaction partitioned between water and ethyl acetate. The aqueous phase was washed with 75 ml of ethyl acetate, and then the combined organic layers were dried over magnesium sulfate. Filtration was followed by solvent removal to afford 150 mg of material, which was carried on to the last step.

The ester from above was dissolved in 1:1 methanol:water and 400 mg of lithium hydroxide was added. The reaction was stirred magnetically under nitrogen for 20 hours at 60° C., then cooled and poured onto 1M NaHSO₄ and extracted three times with ethyl acetate (50 ml×3). The combined organics were washed with saturated brine, then dried over magnesium sulfate. The solvent was removed under reduced pressure, then the crude product (101 mg) was obtained following recrystallization from t-butyl methyl ether/hexanes. Flash chromatography of this material using 1:1 hexanes:ethylacetate with 1% acetic acid followed by another recrystallization using ethyl acetate furnished the final product (80 mg, m.p. 146.4-147.4° C.).

EXAMPLE 43 Preparation of 4′-carboxymethyl MPA (27)

A solution of 3-butyn-1-ol (i) (10.0 g, 143 mmol, Aldrich Chemical Company) was stirred at room temperature in 200 ml dichloromethane. Inidazole (12.6 g, 186 mmol) was added, followed by tert-butyldimethylsilyl chloride (22.6g, 150 mmol) (TBSCl) and the solution stirred for 20 minutes. The reaction was diluted with methylene chloride and washed with water, 1N aqueous HCl solution, brine, and dried over magnesium sulfate. The solvent was removed in vacuo, the residue diluted with hexanes, filtered, and solvent removed in vacuo to give the silyl ether (ii) as an oil, 23.1 g, which was used without further purification.

A solution of the silyl ether (ii) prepared above (8.8 g, 48 mmol) in 200 ml tetrahydrofuran was cooled under an inert atmosphere to −78° C. and a solution of n-BuLi (31.5 ml, 50.5 ml, 1.6M in hexanes) was added over 15 minutes by syringe. The solution was stirred at −78° C. for 1.5 hours, at which time iodomethane (67.7 g, 480 mmol) was added neat and the solution allowed to warm to room temperature overnight. 20 ml 1N aqueous ammonium chloride solution was added, the tetrahydrofuran removed in vacuo, and the aqueous extracted twice with diethylether. The ether solution was washed twice with water, then brine, and dried over magnesium sulfate. The solvent was removed in vacuo to give 8.5 g of (iii) as an oil, which was used without further purification.

A suspension of bis(cyclopentyldienyl)zirconium dichloride (16.0g, 55 mmol) was stirred in 100 ml THF at room temperature under an inert atmosphere. A solution of lithium triethylborohydride (55 mmol, 55 ml, 1M in tetrahydrofuran) was added, and the solution stirred for 1 hour. A solution of the silylether (iii) prepared above (5.0 g, 27 mmol) in 10 ml of tetrahydrofuran was added via a cannula. The reaction was then heated to 50° C. for 1.5 hours, then cooled to 25° C. N-Bromosuccinimide (9.8 g, 55 mmol) was then added, the reaction stirred for 20 min. and then quenched with 50 ml 1N sodium thiosulfate. The mixture was diluted with diethyl ether, filtered through celite, and washed successively with 1 N sodium thiosulfate and brine, then dried over magnesium sulfate. The residue obtained after removing solvents in vacuo was chromatographed over silica gel using hexanes as eluent to give the product vinyl bromide (iv) as an oil (2.1 g), which was carried on to the next step.

A suspension of magnesium (350 mg, 14 mmol) was stirred in a mixture of 20 ml diethyl ether and 5 ml benzene under an inert atmosphere at room temperature. To the mixture was added ethylene dibromide (2.7g, 14.4 mmol, 1.24 ml) over 30 minutes via syringe, keeping the reaction at a gentle reflux. In a separate flask a solution of the vinyl bromide (iv, 3.65 g, 13 mmol) was stirred in 100 ml THF at −78° C. To this solution was added tert-butyllithium (27.4 mmol, 16.1 ml of a 1.7 M solution in hexanes) dropwise over 15 minutes. To the resulting vinyl lithium solution was added the solution of magnesium bromide from above via cannula over 10 minutes, and the solution stirred at−78° C. for 30 minutes to form a vinyl Grignard solution. A solution of the aldehyde (v) [Smith et al., J. Org. Chem. 61, 2236-2241 (1996)] (5.0 g, 14.4 mmol) in 10 ml THF was added to the vinyl Grignard solution via cannula over 5 minutes. The reaction was allowed to warm to 0° C. and stirred for 25 minutes. The reaction was then quenched with 30 ml of 1 N aqueous ammonium chloride solution, extracted with diethyl ether 3 times, and the ethereal solution washed with 1 N aqueous ammonium chloride solution, brine, and dried over magnesium sulfate. The solvent was removed in vacuo and the residue purified by chromatography over silica gel using a gradient of 10% to 20% ethyl acetate in hexanes to give 2.90 g of the allylic alcohol product (vi) as an oil.

A mixture of the allylic alcohol (vi, 2.6 g, 4.7 mmol) was heated in a mixture of trimethylorthoacetate (11.4 g, 12.0 ml, 95 mmol) and pivalic acid (48 mg, 0.5 mmol) at reflux for 11 hours. The reaction was cooled and stirred at room temperature overnight. The reaction was then diluted with diethyl ether, washed with water, saturated aqueous bicarbonate solution, brine, and dried over magnesium sulfate. Solvents were removed in vacuo and the residue chromatographed over silica gel using 15% ethyl acetate in hexanes as eluent to give 1.5 g of the methyl ester product (vii) as an oil.

A solution of the methyl ester (vii, 1.5 g, 2.5 mmol) in 50 ml acetonitrile was stirred at room temperature. A solution of 5 ml of 40% HF solution diluted in 50 ml acetonitrile was added and the reaction stirred for 5 minutes. The solvent was removed in vacuo. The residue was dissolved in diethyl ether, washed 3 times with saturated aqueous bicarbonate, then brine, and dried over magnesium sulfate. Solvents were removed to give 1.0 g of the lactone (viii) as an oil which was used without further purification.

A solution of the lactone (viii, 1.0 g, 2.0 mmol) was dissolved in 10 ml acetone and cooled to 0° C. To the solution was added Jones reagent (1.2 ml, 3.6 mmol, of a 3 M aqueous solution) and the reaction stirred for 45 minutes. The reaction was quenched with 3 ml methanol, diluted with ethyl acetate, washed with 1 N aqueous hydrochloric acid, water 2 times, brine, and dried over magnesium sulfate. Solvents were removed in vacuo and the residue was chromatographed on silica gel using 35% ethyl acetate and 1% acetic acid in hexanes as eluent to give 463 mg of the protected diacid (ix) as an oil.

The protected diacid (ix, 401 mg, 0.8 mmol) was stirred in 10 ml methanol at room temperature. Lithium hydroxide hydrate was dissolved in a solution of 10 ml methanol and 2 ml water and added to the solution of diacid, and the reaction stirred for 3.5 hours. The reaction was quenched with 10 ml 1 N aqueous hydrochloric acid, extracted with ethyl acetate 3 times, the ethyl acetate layers combined and washed with brine. Solvents were removed and the solid so obtained was triturated with a mixture of diethyl ether and hexanes. The resulting solid was recrystallized from methylene chloride/hexanes to give 201 mg of 4′-carboxymethyl MPA (27), m.p.=178° C. 

What is claimed is:
 1. A method for determining an analyte in a sample comprising the steps of: a. combining a sample suspected of containing said analyte with a ligand-inhibitor conjugate comprising a ligand of said analyte and an uncompetitive inhibitor of IMPDH, a receptor specific for said analyte, IMP, NAD and IMPDH under conditions suitable for IMPDH activity, b. monitoring the production of NADH, and c. correlating the production of NADH with the presence or amount of said analyte in said sample.
 2. The method of claim 1, wherein said ligand is selected from the group consisting of drugs, drug derivatives, hormones, polypeptides and oligonucleotides.
 3. The method of claim 2, wherein said uncompetitive inhibitor is mycophenolic acid or a mycophenolic acid analog.
 4. The method of claim 3, wherein said analyte is mycophenolic acid.
 5. The method of claim 3, wherein said analyte is thyroxine.
 6. The method of claim 3, wherein said analyte is digoxigenin.
 7. The method of claim 3, wherein said analyte is theophylline.
 8. A reagent for determining an analyte in a sample comprising a ligand-inhibitor conjugate comprising a ligand of said analyte and an uncompetitive inhibitor of IMPDH.
 9. The reagent of claim 8, wherein said uncompetitive inhibitor is mycophenolic acid or a mycophenolic acid analog.
 10. The reagent of claim 8, wherein said analyte is selected from the group consisting of drugs, drug derivatives, hormones, polypeptides and oligonucleotides.
 11. The reagent of claim 10, wherein said analyte is mycophenolic acid.
 12. The reagent of claim 10, wherein said analyte is thyroxine.
 13. The reagent of claim 10, wherein said analyte is digoxigenin.
 14. The reagent of claim 10, wherein said analyte is theophylline.
 15. A kit for conducting an assay for the determination of an analyte in a sample comprising in packaged combination: a. a first reagent comprising NAD, and b. a second reagent comprising IMP, IMPDH and a ligand-inhibitor conjugate comprising a ligand of said analyte and an uncompetitive inhibitor of IMPDH, wherein said first or said second reagent further comprises a receptor specific for said analyte.
 16. The kit of claim 15, said kit further comprising a calibration reagent comprising a known amount of said analyte.
 17. The kit of claim 15, wherein said uncompetitive inhibitor is mycophenolic acid or a mycophenolic acid analog.
 18. The kit of claim 15, wherein said analyte is selected from the group consisting of drugs, drug derivatives, hormones, polypeptides and oligonucleotides.
 19. The kit of claim 18, wherein said analyte is mycophenolic acid.
 20. The kit of claim 18, wherein said analyte is thyroxine.
 21. The kit of claim 18, wherein said analyte is digoxigenin. 